Bisphosphoglycerate mutase (BPGM)
created by Brett Moody
Bisphosphoglycerate mutase (BPGM) from Homo sapiens (PDB ID=2H4Z) is a trifunctional enzyme that's primary function is the synthesis of 2,3-bisphosphoglycerate (2,3-PBG) from 1,3-bisphosphoglycerate (1, 2). BPGM's only ligand is 2,3-BPG (14). The amount of 2,3-BPG in the blood stream is regulated by BPGM. 2,3-BPG is the allosteric inhibitor of hemoglobin that shifts the equilibrium between the oxygenated and deoxygenated form (2). 2,3-BPG does this by stabilizing the deoxygenated form and inducing the transport of oxygen to tissues (8). The presence of 2,3-BPG is shown to facilitate the polymerization of deoxygenated hemoglobin mutants (2). These mutants can yield deformed erythrocytes and cause vaso-occlusive complications (blockage of a blood vessels (4)), both of which are characteristics of the disease sickle-cell anemia. Therefore, controlling the level of 2,3-BPG in the blood stream has important implications for the treatment of such diseases (2). Because BPGM can also function as a phosphatase by catalyzing the hydrolysis of 2,3-BPG to a phosphoglycerate, one therapeutic approach is to increase the phosphatase activity of BPGM (2). BPGM also functions as a mutase by catalyzing the interconversion between 2- and 3-phosphoglycerate (2). The molecular weight of BPGM is 62122.67 Da and the isoelectric point (pI) is 6.29 (3). The molecular weight and isoelctric point were calculated using the Expasy bioinformatics search. The isoelectric point is the pH at which the protein carries no net charge (12).
The structure of bisphosphoglycerate mutase is related to its function as an enzyme. BPGM is a homodimer. A homodimer is a protein dimer that consists of two identical monomers (13). Each monomer in BPGM has multiple α-helices and β-strand domains. The dimer is formed between the surface of a parallel β-strand and an α-helix of each monomer. A salt bridge between Lys-29 and Glu-72 as well as several other hydrogen bonds help to stabilize the dimer. The core of the protein consists of a β-sheet formed by five parallel running β-strands and one anti-parallel running β-strand. The β-sheet is flanked by six α-helices. The side chains of the Ile-64, Trp-68, Leu-69, Leu-71, and Val-81 residues form the hydrophobic core (2). The C-terminal possibly forms an α-helix that helps accommodate the substrate to the active site (2).
The C-terminal contains hydrogen bonds between Lys-246 and Glu-249 as well as Glu-33 and Lys-247. These bonds may stabilize and fix the C-terminal to the active site pocket (2). The C-terminal tail contains many important residues that are necessary for the protein's function. Deletion of the last 7 residues yields a completely defective protein that cannot carry out any of its three catalytic activities (synthase, mutase, phosphatase) (2). PBGM contains only one active site. The residues His-11, His-188, Arg-10, and Arg-62 are located at the bottom of the active site pocket. The opening of the active site has Lys-18, Asn-20, Arg-100, and Arg-116 on one side and Ile-208, Asn-209, and Thr-211 on the other. The presence of bases at the active site gives a "highly positive electrostatic potential with negatively charged substrates" (2). Electrostatic potential is potential energy resulting from charge differences and similarities (12).
When BPGM binds with its substrate, residues 99-122 move toward the active site by rotating towards the core of the protein. This places Arg-100, Arg-116, and Arg-117 into the active site. Meanwhile, C-terminal residues 236-250 shift along the C-terminal helix and residues 251-256 stretch forward closing the active site. His-188 hydrogen bonds to His-11 to help His-11 attain its correct position for the reaction (2). His-11 is a residue that is critical for protein structure and/or its biological function, also known as a critical residue (9). His-11 forms a line with the substrate's central phosphorus atom and C-2 oxygen atom in order to phosphorylate/dephosphorylate the substrate.
E. Coli cofactor-dependent Phosphoglycerate mutase complexed with vanadate (pdb ID= = 1E59) is a cofactor dependent phosphoglycerate mutase, also referred to as a dPGM, that has an approximate sequence similarity of 50% to BPGM (2). The Bioinformatics search databases DALI and BLAST were used to find similarities between proteins. DALI compares the tertiary structure of proteins by using a "sum-of-pairs method that compares intramolecular distances" (10). The similarities between the two proteins is reflected in a Z-score. A Z-score above 2 indicates that the proteins have significant similarities (10). PSI-BLAST is used to find primary structure similarities. PSI-BLAST calculates an E value based on the total sequence homology and by assigning gaps. The lower the E value, the more similar the protein's primary structures are. An E value that is below 0.05 is considered significant (11). The results of the DALI (Z-score = 34.5 and RMSD = 1.8) and PSI-BLAST (E = 6e-167) operations indicate that BPGM and dPGM have similar tertiary and primary structure (6, 7). Both proteins have a similar α/β-fold and yield a root mean square distance of 0.74 Å when superimposed on each other in the BPGM residue regions: 4-11, 25-97, and 157-199. This indicates a strong structural homology (2). The most significant differences between the two proteins are seen in the active site, C-terminal, and backbone. In BPGM, Gly-14 orients Glu-13 and Phe-22 in a way that prevents the active site from crowding. Gly-14 functions as a critical residue only in BPGM (in the dPGM it is replaced by Ser-14) (2). The dPGM C-terminal has 9 less residues than BPGM. The C-terminal plays an important role in the enzymatic activity. The loss of these 9 residues may play a role in each of these protein's preferences for substrates (2). A four-residue insertion in dPGM occurs at residues 136, 137, 143, and 144. This forms an additional α-helix from residues 133-138. Because this additional α-helix is far from the active site, the structural difference does not effect the enzymatic function. Additional hydrogen bonds seen only in BPGM are present between Ser-63 and Ser-186, as well as Ser-192 and His-188 and Gly-189. These exist near the active site and may help to stabilize BPGM's structure (2).
When functioning as a mutase, BPGM's critical residue Glu-89 donates/recieves a proton during the phosphotransfer step (2). However unlike dPGM, in BPGM the residue Glu-13 is pointed towards the active site and forms a hydrogen bond with Glu-89. This reduces Glu-89's ability to donate/receive protons and is one possible reason for the lower mutase activity of BGPM (2).